W=WPRE sequence; L=long version of promoter

Background on pHAGE: A third generation lentiviral vector

The lentiviral system we employ is based on an HIV-1-based backbone, named ‘pHAGE’ (standing for plasmid HIV-1 Alex Gustavo George Enhanced) originally developed in the laboratory of Dr. Richard C. Mulligan at Harvard Medical School. Dr. Kotton worked with the original pHAGE backbone during his postdoctoral fellowship in the Mulligan laboratory and the backbone has since been adapted to express the transgenes indicated on this page.

To summarize, the third generation lentiviral vectors employed in our studies are typically pseudotyped with a VSV-G envelope that allows stability and tropism for cells of multiple species including mice. The engineered virus is replication incompetent and has a self-inactivating viral promoter (deleted U3). This ensures that, after integration in the target cell genome, only the internal promoter of interest is active and no further viral replication or infection occurs. A woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and cPPT is included in each vector to increase gene expression levels in transduced cells.

How to Clone into the pHAGE vector backbone

Cloning into the pHAGE vector backbone is relatively simple. We have single promoter versions as well as double promoter and IRES-containing versions designed to accomplish single cDNA or dual cDNA vector expression. The plasmid backbone is modular with NotI and BamH1 sites flanking the first gene expression cassette (allowing any cDNA to be cloned into any pHAGE vector after attaching 5′ NotI and 3’BamH1 sites to your cDNA of interest). To design primers to add these sites to your cDNA, see the suggestions below. Also please refer to our CReM protocols page for a free downloadable .pdf version of this protocol as well as many other useful lenti and stem cell protocols.

Primer design for cloning genes into the lenti backbone:

NB: Not1= GCGGCCGC and Kozak consensus sequence= GCCA/TGCCATGG; so a 5’ oligo for PCR of a gene to clone into position one should add NNN-GCGGCCGCC in front of the ATG start codon for that gene thus adding the Kozak consensus sequence with embedded Not1 site onto the 5’ end of the gene! (NNN= three random nucleotides {e.g. TTT} in front of the restriction site to allow Not1 enzyme to cut efficiently.

2nd position:promoter: BamH1/Nde1
2nd gene CDS: Nde1/Cla1

NB: Nde1 enzyme requires 7 base pairs in after the restriction sequence for efficient cutting. Also beware CMV promoter has an internal Nde1 site so this site will not be unique when working with CMV containing constructs. Also ClaI in certain context gets dam methylated (see other protocol titled ClaI dam methylation).

How to design primers using Vector NTI: Enter sequence of your gene. Start with the 5’ (upper) primer. Paste about 40 base pairs of the 5’ leading end of your sequence into the oligo list. Add the restriction site in front of your sequence with extra leading base pairs (e.g 3 extra base pairs for not1 to allow cutting; don’t forget to add kozak consensus sequence in front of ATG for genes). Save this sequence in the oligo list as’ oligo.

Next analyze and trim the oligo: In the oligo menu click analyze. Remove the extra (non- complementary) leading sequence that you are going to add to the 5’ end of the gene as this will not bind to anything on the first PCR cycle: click analyze. Begin trimming bases away from the 3’ end of the sequence until the Tm is about 60 degrees. Paste back the 5’ leading sequence that you want to add to the gene during PCR. Save this and analyze it for hairpins, dimmers etc.